To date, the majority of chromatin studies on brain are focused on the regulation of DNA methylation and post-translational histone modifications. However, replication-independent nucleosome remodeling and chromatin assembly, a mechanism associated with the process of gene expression, is likely to be of crucial importance for neurons and other postmitotic cells. Presently, next to nothing is known about its regulation in mature CNS. This is mainly due to methodological barriers, due to the difficulty to selectively sort neuronal chromatin from brain tissue and the lack of reliable antibodies to study the exclusive substrate for replication independent deposition, which is the histone H3 variant H3.3. The goal of this exploratory proposal is to overcome these critical shortcomings for brain-related chromatin assays. Specifically, we propose to develop a toolbox that includes (i) a transgenic mouse line expressing green fluorescent protein (GFP)-tagged histone H2B in a select set of forebrain neurons, which is complemented by (ii) adeno-associated virus (AAV)-based delivery of an expression cassette for FLAG/HA-epitope-tagged H3.3 under control of the c-fos promoter and (iii) acute challenge with dopamine D1 receptor agonist and D2-like antagonists, a well known trigger for early response gene expression and other transcriptional activity in prefrontal cortex and striatum. The proposed experiments will, for the first time, explore the phenomenon of replication-independent chromatin assembly and histone H3.3 deposition in neuronal nuclei of adult mice on a genome-wide scale, by combining chromatin immunoprecipitation techniques with massively parallel sequencing (Chip-seq). These pioneering studies will provide first insights into the role of replication-independent histone deposition in the mature nervous system, including the potential association with neuronal gene expression in response to changes in dopaminergic input. PUBLIC HEALTH RELEVANCE: The majority of common neurological and major psychiatric diseases (Alzheimer's, schizophrenia, autism, depression, druga, just to name a few examples) are thought to involve a combination of genetic and non-genetic factors. The latter includes "epigenetic" mechanisms, which typically are defined by a variety of chemical and molecular modifications of the genomic DNA and of the surrounding chromatin, without a change in the DNA sequence. One interesting layer of epigenetic regulation involves the exchange of certain histones (a type of protein closely attached to the genomic DNA) during the process of gene expression. The role of this mechanism, also called replication independent nucleosome exchange and histone deposition, is at present totally unexplored in the normal and diseased brain. The goal of this grant application is to overcome this critical shortcoming for brain-related epigenetic studies. Specifically, we propose to generate mice in order to assay replication independent histone deposition in specific types of nerve cells in the mouse brain.